The present invention relates generally to the field of detecting and quantifying apoptosis. More particularly, the present invention relates to antibodies to the newly formed amino terminus resulting from cleavage of proteins during the process of apoptosis and to the use of such antibodies in detecting apoptosis in cells undergoing apoptosis or in cells that have undergone apoptosis.
Most eukaryotic cells have the ability to self-destruct by activation of an intrinsic cellular suicide program referred to as programmed cell death or apoptosis. The process of apoptosis involves a cascade of cytoplasmic and nuclear events that result in a series of morphological changes, and eventually cause the demise of the cell. Apoptosis is characterized by distinct biochemical and morphological changes exhibited by cells undergoing programmed cell death, including DNA fragmentation, plasma membrane blebbing, and cell volume shrinkage. At the molecular level, activation of one or more aspartate-specific, cysteine proteases (caspases) is proposed to be the critical signal required to carry out apoptotic cell death (Yang et al., American Journal of Pathology, 152(2):379-389, 1998).
The caspases, also known as ICE (IL-1 xcex2-converting enzyme)-like proteases, can be divided into three subclasses: ICE/CED3 family, CPP32/Yama family and the Ich/Nedd2 family (Duan et al., J. Biol. Chem., 271:1621-1625, 1996). All family members share a high level of amino acid sequence homology with ICE, and contain a conserved QACRG pentapeptide in which the cysteine participates in catalysis (Nicholson, Nature Biotech., 14:297-301, 1996). Furthermore, all of these proteases are reported to require an aspartic acid residue at the substrate P1 position (Jxc3xa4nicke et al., The EMBO J., 15(24):6969-6978, 1996).
CPP32 (Caspase 3) has been identified as one of the proteases that cleaves poly(ADP-ribose) polymerase (PARP) (Schlegel et al., J. Biol. Chem., 271:1841-1844, 1996; Nicholson et al., Nature, 376:37-42, 1995). PARP is one of the enzymes associated with DNA repair. Cleavage of the approximately 116 kilodaltons (xe2x80x9ckdxe2x80x9d) PARP protein into fragments of about 89 kd and about 27 kd has been reported to contribute to the DNA fragmentation that is characteristic of apoptosis (Kayalar et al., Proc. Natl. Acad. Sci. USA, 93:2234-2238, 1996). Therefore, the identification of the about 89 kd or the about 27 kd fragments resulting from the cleavage of PARP within a cell is an indication that the cell is undergoing or has undergone apoptosis.
Proteins consist of macromolecules of amino acids linked by peptide bonds, to form polypeptide chains. Each amino acid in the chain consists of a carbon atom to which are attached four different groups (xe2x80x94R, xe2x80x94H, xe2x80x94NH2, and xe2x80x94CO2H), wherein the identity of R varies from one amino acid to another. The peptide bond links each amino acid to the next amino acid in the chain through a covalent bond formed between the xe2x80x94CO2H group of one amino acid and the xe2x80x94NH2 group of the next amino acid, with H2O a byproduct of the reaction. Every polypeptide chain has two terminal amino acid residues, one at each end of the chain. The end of the chain with a xe2x80x94CO2H group which has not been linked to another amino acid is referred to as the xe2x80x9ccarboxy terminusxe2x80x9d or xe2x80x9cC-terminusxe2x80x9d. The end of the chain with a xe2x80x94NH2 group which has not been linked to another amino acid is referred to as an xe2x80x9camino terminusxe2x80x9d or xe2x80x9cN-terminusxe2x80x9d. When an enzyme such as caspase cleaves PARP, it breaks a peptide bond in a polypeptide chain of the protein, creating one fragment with a new carboxy terminus and another fragment with a new amino terminus.
One reference, WO 98/21590, describes methods of detecting apoptosis by using antibodies that bind to the amino acids at the newly created carboxy termini of polypeptides generated by the cleavage of proteins by the caspase family of proteases. Because caspases cleave immediately carboxy-terminal of a characteristic four amino acid recognition site, the newly created carboxy terminus of a caspase cleaved protein consists of the last amino acid of the recognition site. WO 98/21590 describes the production of antibodies following immunization of rabbits with a polypeptide comprising a caspase recognition site (GDEVD) at its carboxy terminus. Although the antibodies of WO 98/21590 appear to recognize a cleaved fragment of PARP, these antibodies were cross-reactive with other proteins as well. This cross-reactivity is likely to result in inaccurate determinations of apoptosis in cells or cellular lysates.
In another attempt to produce antibodies that are specific to apoptotic fragments of PARP, Sallmann et al., Biochem. Cell Biol., 75:451-456, (1997) immunized rabbits with synthetic polypeptides corresponding to the newly created carboxy terminus and amino terminus of PARP that are formed following cleavage of PARP by a caspase. Although the polyclonal antibodies produced by Sallmann et al. were able to distinguish between the two (carboxy terminal and amino terminal) apoptotic fragments of PARP, the antibodies were not able to distinguish between the cleaved fragment and uncleaved PARP. Therefore, the antibodies produced by Sallmann et al. are not specific to epitopes produced in apoptotic cells because they are immunoreactive with the uncleaved PARP present in non-apoptotic cells.
Therefore, there is a need for antibodies that are specifically able to distinguish apoptotic events in cells. These antibodies will enable more accurate results in methods for detecting apoptosis. Because apoptosis, or the inability of cells to undergo apoptosis, is associated with a number of disorders and diseases including cancer, neurodegeneration, autoimmunity, heart disease and others (reviewed in Hetts, JAMA, 279(4):300-307, 1998), improved methods of detecting apoptosis will provide a better understanding of these diseases and will be useful in screening potential therapeutic agents that may induce or prevent apoptosis.
The present invention seeks to overcome limitations in the prior art by providing methods and compositions related to antibodies that recognize specific epitopes that are indicators of apoptosis. Antibodies were produced that are specific to a new epitope (neoepitope) formed in apoptotic cells. The neoepitope is a product of cleavage of a protein at a specific cleavage site by a protease during apoptosis.
The present invention involves the use of antibodies raised against a neoepitope which comprises a polypeptide sequence which is homologous to only one end of one of the fragments of a protein cleaved during apoptosis. Every protein consists of a chain of amino acids. Each amino acid covalently linked to the next amino acid in the chain through a peptide bond. A peptide bond links the amine residue of one amino acid with the carboxyl residue of the next amino acid in the chain. The resulting chain has an unattached amine residue at one end, and an unattached carboxy residue at the other end. When the protein is cleaved, and the chain of amino acids broken at that point, two fragments of the protein are formed, one with a new carboxy terminus, and the other with a new terminal amine. In the present invention, the neoepitope comprises the new amino terminus at the beginning of the carboxy terminal fragment.
By immunizing animals with a polypeptide comprising a sequence of the amino acids of the newly formed amino terminus of a fragment of a protein formed by cleavage of the protein during apoptosis, antibodies were isolated that recognized the cleaved form of the protein but failed to recognize the uncleaved form. This result is surprising because attempts by others to produce specific antibodies to a new amino terminus of PARP formed after cleavage by a caspase failed because the antibodies were still immunoreactive with the uncleaved protein (Sallmann et al., Biochem. Cell Ciol., 75:451-456, 1997). Furthermore, the isolated antibodies of the present invention show particular utility in methods of detecting apoptosis.
The present invention includes compositions comprising an isolated antibody that is immunoreactive with a neoepitope produced in a cell undergoing apoptosis. As used herein, the term immunoreactive means that the antibody is capable of binding the antigen with an affinity that is indicative of an immune reaction to the antigen. Such affinities are well known to those of skill in the art and include affinities of 105 to 1014 Mxe2x88x921. Methods of determining the affinity of an antibody composition are described in Day, Advanced Immunochemistry, (2nd edition) Wiley-Liss, New York, N.Y. (1990).
Although binding the neoepitope is an important aspect of the present invention, it is also important that the antibody fails to bind the uncleaved protein. This ability to distinguish between the cleaved and uncleaved form of a protein gives the antibody its novel specificity to detect apoptotic cells, as the neoepitope is at a detectable level in apoptotic cells but below a detectable level in non-apoptotic cells.
The antibody may be a polyclonal or monoclonal antibody and recognizes the new amino terminus of poly(ADP-ribose) polymerase (PARP) formed by cleavage of the protein by a caspase. A polypeptide comprising the amino acid sequence of FIG. 1 (SEQ ID NO:2), when injected into a rabbit, was able to cause production of antibodies that bound specifically to the cleaved form of PARP while failing to bind the uncleaved form.
Although the methods of the present invention may be employed with a number of different proteases, the inventor has found that targets of the caspase family of proteases are particularly useful. The caspase family of proteases include ICE (caspase 1), caspase 3, caspase 7, and caspase 8, and are reviewed in Nicholson, et al., TIBS, 22(8):299-306 (1997) and Villa et al., TIBS, 22:388-393 (1997). In preferred embodiments, the neoepitope is the new amino terminus of a protein cleaved by caspase 7 or caspase 3.
An antibody of the present invention may be immunoreactive with an epitope in apoptotic cells from a number of species including chicken, bovine, murine, feline, canine, rat, equine, opine, and primate species including human. In preferred embodiments, the antibody is immunoreactive with an epitope in apoptotic cells of a mammal, preferably human.
The apoptotic cell may be a cell of essentially any type known to be capable of the process of apoptotis including heart, lung, skeletal muscle, neuronal, liver, kidney, pancreas, epithelial, or blood cell. In preferred embodiments, the blood cell is a leukemia cell such as HL-60.
The antibodies of the present invention may be used in methods of detecting apoptosis in a cell or group of cells. The methods disclosed herein allow the determination of apoptosis in a biological sample comprising an individual cell or a group of cells. As used herein a group of cells may be any collection of more than one cell such as a blood sample, tissue sample, biopsy, or tissue culture. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide.
In some embodiments, the methods of detecting apoptosis in a cell or group of cells comprise obtaining a protein sample from the cell or group of cells, contacting the protein sample with an antibody which is immunoreactive with a neoepitope in apoptotic cells, and screening the cell or group of cells to detect any of the antibody bound to the neoepitope. Detecting the antibody bound to the neoepitope in the sample is indicative of apoptosis in the cell or group of cells, whereas failing to detect the antibody bound to the neoepitope is indicative of lack of apoptosis in a cell or group of cells. Such methods include immunoassays such as Western blots, Enzyme Linked Immunosorbent Assay (xe2x80x9cELISAxe2x80x9d), cell-based ELISA, filter-binding ELISA, inhibition ELISA, sandwich ELISA, immunostaining, immunoprecipitations, slot or dot blots, radioimmunoassays, scintillation proximity assays, Ouchterlony analysis, and fluorescent immunoassays. Some of the above methods require the cells to be lysed or processed to isolate proteins therefrom prior to the detection step.
Other methods of the present invention for detecting apoptosis in cells do not require the step of acquiring a protein sample or lysate, but rather detect apoptosis in the cells or tissues themselves. Such methods include immunohistochemistry, immunocytochemistry, and flow cytometry.
In some embodiments, the antibody of the present invention further comprises a label. A label is a molecule or substance that is attached to the antibody that facilitates detection of the presence of the antibody. Labels are well known to those of skill in the art and include, but are not limited to, haptens such as biotin or nitro-iodo-phenyl, radioactive isotopes such as 125I, 3H, 14C, 32P, or 35S, enzymes such as alkaline phosphatase, horseradish peroxidase, xcex2-galactosidase, or luciferase, and fluorescent or luminescent molecules, such as fluorescein, rhodamine, phycoerythrin, Texas red, green flourescent protein or derivatives thereof.
Antibodies of the present invention comprising labels are particularly useful in methods of detecting apoptosis in a cell, group of cells, cell sample, or sample of a group of cells. In other embodiments, the presence of an antibody of the present invention is detected by the use of a secondary antibody comprising a label.
Another embodiment of the present invention is a method of producing an antibody immunoreactive with a neoepitope produced in a cell undergoing apoptosis. This method comprises the steps of: (a) obtaining a polypeptide comprising an amino acid sequence corresponding with the amino terminus produced by cleavage of a protein by a protease during apoptosis; and (b) administering the polypeptide to an animal under conditions suitable to provoke an immune response thereby producing antibodies to the polypeptide. In preferred embodiments of this last method, antibodies obtained by such a method are removed from the animal, or in the case of a chicken are removed from the egg, and tested to ensure that they are not immunoreactive with the uncleaved protein.
The methods of the present invention may be used for diagnosing a disease, disorder, or condition associated with cell apoptosis. Such methods comprise contacting a cell, tissue, group of cells, or samples thereof, with an antibody of the present invention and detecting, or failing to detect, the antibody bound to the neoepitope. Detecting the antibody bound to the neoepitope is indicative of apoptosis, whereas failing to detect the antibody bound to the neoepitope suggests apoptosis is not present in the cell, tissue, group of cells, or samples thereof.
In yet another embodiment, the present invention provides methods of screening compounds to identify inhibitors of apoptosis. These methods comprise exposing a sample of cells to conditions known to activate apoptosis in the cells, contacting the sample with a test or candidate compound, contacting the sample with an antibody of the present invention, and quantifying or detecting the level of antibody bound to the neoepitope in the sample. In preferred embodiments, a second sample is induced into apoptosis and is subjected to the same steps as the first sample except the second sample is not contacted with the test compound. The use of the untreated (by the test compound) second sample allows one to compare the level of antibody bound to the neoepitope in cells treated with the test compound, versus antibody levels in cells not treated with the test compound. The test compound is said to inhibit apoptosis if the level of the antibody in the first sample is less than the level of the antibody in the second sample.
In another embodiment, the present invention provides methods for screening compounds to identify stimulators or inducers of apoptosis. Such methods comprise contacting a sample of cells with a test compound, contacting the sample with an antibody of the present invention, and quantifying or detecting the level of antibody bound to the neoepitope in the sample. In preferred embodiments, a second sample is subjected to the same steps as the first sample except the second sample is not contacted with the test compound. The use of the untreated second sample allows one to compare the level of antibody bound to the neoepitope in cells treated with the test compound versus cells not treated with the test compound. The test compound induces apoptosis if the level of the antibody in the first sample is greater than the level of the antibody in the second sample.
Also provided by the present invention are kits for detecting apoptosis. Specifically, the kits are for detecting apoptosis-generated protein fragments in a sample. Such kits comprise an antibody of the present invention. In some embodiments, the antibody of the present invention comprises a label. In other embodiments, the antibody of the present invention is not labeled, but the kit further comprises a labeled secondary antibody that is immunoreactive with the antibody of the present invention.
Other reagents that the kits of the present invention optionally comprise antibodies immunoreactive with surface antigens (such as CD4, CD8, TCR, B220, Fas), antibodies immunoreactive with a proliferation or other marker antigens (such as p21, p53, Rb, PCNA, Ki-67, etc.), or reagents for the TUNEL (TdT-mediated dUTP Nick-End Labeling) reaction (Promega Corporation, Cat. No. #G3250, G7360).
Also provided, are methods of producing an antibody immunoreactive with a neoepitope comprising the amino terminus produced by cleavage of a protein by a protease during apoptosis, but not immunoreactive with the uncleaved protein. Such methods comprise obtaining a polypeptide comprising the amino terminus produced by cleavage of a protein by a protease during apoptosis, administering the polypeptide to an animal to elicit antibody production against the polypeptide, and collecting the antibodies from the animal by methods known to those of skill in the art.
As used herein, xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d are defined to mean one or more.